Pertussis, the most responsible agent of which is the gram-negative bacterium Bordetella pertussis, remains a current infectious disease throughout the world. More than 50 millions cases are declared each year and a mortality estimation is 500 000 deaths per year, mainly infants. Mortality is for example high in the developing countries where vaccination is not sufficient and pertussis represents one of the major causes of infantile mortality. The advent of cellular vaccines consisting in chemically or thermally inactivated B. pertussis has led to a spectacular reduction of pertussis occurrence. However, limitations related to their use have appeared during these last two decades. Various reports have thus put forward the hypothesis of a relationship between the administration of the cellular vaccine against pertussis and some serious side-effects.
Moreover, the need to administrate various vaccinal doses to obtain a maximum protection, the variability of efficiency of the different batches of vaccines and the resurgence of the disease in adults due to a weakening of post-vaccinal immunity during the period of the post-vaccinal immunity in the absence of late re-vaccinations are as many inconveniences that have led to the development of cellular vaccines. However, the protective antigens comprising those new generation vaccines, generally defined as more efficient, more secure and more immunogenic than cellular vaccines, have not been clearly defined yet and multiple injections are still necessary. Moreover, serological correlations with the protection of children have not still been determined. Finally, the production cost of these vaccines has been found higher than this of the prior ones, which represents an important problem for developing countries that are most demanding.
On the other hand, it has been shown that the natural infection with B. pertusiss leads to a long-term protection, whereas the one induced by vaccination had a limited time (Bass, J. W. et al. 1987 Pediatr. Infect. Dis. J. 6:141-144, Jenkinson, D. 1988, Br. Med. J. 296:612-614). The reasons for this difference are not clear, showing the lack of knowledge relating to immunizing mechanisms involved in each case. One of the potential explanations is based upon a better induction of the immunologic memory at the respiratory tract after an infection, compared to the one obtained after a parenteral immunization. Moreover, a Th1-type cellular response T, which could play an important part in the protective immunity, is induced through the natural infection, whereas immunization with the cellular vaccine shows rather a strong response Th2 (Mills, K. H. G. et al. 1983, J. Med. Microbiol. 39:163-164). Finally, the infection stimulates IgA production against B. pertussis antigens in the serum and the secretions, whereas these antibodies are not frequently detected on vaccinated individuals (Shahin, R. et al. 1992, in J. E. Ciardi et al. Eds Genetically Engineered Vaccines, Plenum Press N.Y.).
All those observations led to the belief that a long-term protection against pertussis would be better obtained through a vaccination with attenuated living bacteria administrated at the level of the respiratory mucosas, which are the natural openings during the infection by B. pertussis. Prior work has shown that immunization of mice either with spontaneous attenuated living strains (Vesselinova-Jenkins, C. K., 1985. Dev. Biol. Stand. 61:517-524) or aroA strains (Roberts, F. et al. 1990. Infect. Immun. 58:732-739) of B. pertussis protects against a second infection. However, such strains are inconvenient in that they are badly characterized and hold a certain toxicity, or have lost to a great extent their colonizing capacity, and thus require numerous administrations to induce an efficient protective immunity.
The B. pertussis toxin is an oligomer protein formed with five sub-units so-called S1 to S5. It may be shared into two great domains so-called protomer A (consisting in S1) and oligomer B (consisting in S2 to S5). The oligomer B is responsible for the toxin interaction with its receptors at the surface of the target cells and the protomer A (or S1) is injected into the target cell and expresses therein an ADP-ribosyltransferase enzyme activity (Tamura et al., Biochemistry 21, 5516-5522, 1962). The genes coding for the five sub-units have been cloned and sequenced (Locht and Keith, Science 232, 1258-1264, 1986) and the crystal structure has been established (Stein et al., Structure 2, 45-47, 1994). Various mutations in the toxin gene have been described as being responsible for the genetic inactivation of this molecule. These mutations may be found in S1 gene (review, see Locht and Antoine, Biochimie 77, 333-340, 1995) and/or in the genes coding for the oligomer B (see for example Lobet et al. J. Exp. Med. 177, 79-87, 1993). The toxin may be put in evidence through an immunologic activity with polyclonal or monoclonal antibodies, such as for example the antibody lB7 mentioned thereafter or through its biological activity, such as the activity on the cells xe2x80x9cChinese Hamster Ovaryxe2x80x9d as described by Hewlett et al. (Infect. Immun. 40, 1198-1203, 1983).
Filamentous hemagglutinin (FHA) is the major adhesion produced and secreted by B. pertussis (review, see Locht et al. Mol. Microbiol., 9, 653-660, 1993). The structure gene of the FHA, so-called fhaB, has been clond (Brown, D. R. et al. 1987. Infect. Immun. 55:154-161; Relman, D. A. et al. 1989. Proc. Natl. Acad. Sci. USA. 86:2637-2641; Delisse-Gathoye, A.M. et al. 1990. Infect. Immun. 58:2895-2905) and codes for a precursor (FHAB) of about 367 kDa. The mature form (220 kDa) corresponds to the N-terminal two thirds of FHAB and has a structure so-called xe2x80x9chair pinxe2x80x9d (Makhov et al., J. Mol. Biol. 241, 110-124, 1994). Although the C-terminal part of FHAB is not present under the mature form, it has interesting characteristics such as various regions known to be rich in proline and an RGD site. Moreover, this C-terminal part of the precursor appears to play an important part in FHA secretion. Downwards the N-terminal region of the FHA two repetitive regions, respectively A and B, are located, followed by a proteolysis-sensitive site and by a RGD sequence. The FHAB maturation site is located at about 1000 amino-acids downwards this RGD sequence. The FHAB N-terminal domain plays an essential part in the secretion of the mature protein since a phase deletion of this region appears to inhibit totally the FHA biogenesis. The FHA secretion depends upon a minor protein so-called-FhaC present at the level of the external membrane of B. pertussis. This protein is coded by the gene FhaC located downwards FhaB and separated from this latter by three other genes, fimB, fimC and fimD, coding for the fimbriae. An FHA secretion mechanism involving cleavage and then modification of the N-terminal part of FHAB, followed by interaction between this region and the minor protein FHAC, have been proposed.
The various FHA binding mechanisms allow the B. pertussis to adhere to numerous types of eukaryote cells. Although the B. pertussis toxin is involved in the adherence of the bacterium with the epithelial cells of the respiratory tract, the FHA plays a major part in such an interaction. The FHA can interact with glycoconjugates and the carbohydrate recognition domain has been identified in the FHA region limited by the amino-acids 1141 to 1279 which would correspond to the buckle of the xe2x80x9chair-pinxe2x80x9d structure which already contains the RGB site in positions 1097-1099. The FHA is also the major adhesin involved in the adherence mechanism of B. pertussis with the unciliated epithelial cells. An activity of binding this molecule to the heparin sulphated glycosaminoglycan, present in non negligible amount in the bronchial mucus as well as in the extracellular matrix and at the surface of numerous epithelial cells is responsible for the bacterium adherence with these cells. Recent studies make obvious a specific heparin binding site located in the N-terminal region of the FHA, probably in the region comprised between the amino-acids 442 and 863 (Hannah et al. Infect. Immun., 62, 5010-5019, 1994).
Besides this adherence activity on the ciliated and unciliated epithelial cells, FHA is able to bind specifically with the CR3 (CD11b/CD18, aMb2) present on the macrophages and monocytes (Relman et al. Cell, 61, 1375-1382, 1990). This binding of B. pertussis, via the FHA, with the CR3 allows for the internalization of these bacteria in the macrophages through a means generating no oxidative catabolism.
Due to its interest in the adherence of B. pertussis with various cell types, the FHA has been considered as a major antigen for vaccination against pertussis. Administrating a cellular vaccine to mice or children leads to the production of anti-FHA antibodies in their serum. Moreover, vaccinating 18 month old children with a cellular vaccine induces high rates of anti-FHA antibodies as well as specific cellular response T. These immune characteristics are found in convalescent people after a pertussis. 
However, the isotopic profile of the anti-FHA response in children having received the cellular vaccine differs from the one of individuals attacked by pertussis. Whereas the specific serous immunoglobulin G rate (IgG) is similar, the anti-FHA IgA2 response is more important in sick people than in vaccinated people. This difference is still more marked at the level of nasopharyngeal secretions and saliva, where an increase of the anti-FHA IgA rate is observed in most of the patients various weeks after the beginning of the symptoms. Similarly, high anti-FHA IgA rates are found in the nasal secretions of mice infected with the B. pertussis virulent strain in a period up to 26 weeks after the infection. An immune response both IgAs and IgG against the FHA may also be obtained in the respiratory tract of mice after an intranasal vaccination with purified FHA.
Potentially protective epitopes of cells B and T have been localized on the FHA. An immunodominant region has been thus determined in the C-terminal region of the mature FHA. It is recognized by the murine cells B and by the human lymphocytes T CD4+ which also recognize the N-terminal part of the FHA.
Recombinant fusion proteins comprising one part of the FHA and heterologous peptides have been described in the French application FR 94 04661 (published under the No. 2 718 750) (INSTITUT PASTEUR, INSTITUT PASTEUR DE LILLE, INSERM). In this application, recombinant DNAs comprising a sequence coding for a heterologous polypeptide, fused in the same reading frame with a sequence coding for at least a part of the FHA precursor, are prepared and expressed in cell cultures, particularly Bordetella detoxified or attenuated cultures.
This invention relates to a Bordetella strain deficient in the production of toxins and expressing a hybrid protein.
It also relates to liposomes comprising at least a heterologous protein and at least one part of the FHA (filamentous hemagglutinin) protein.
It also relates to the immunoprophylactic and/or therapeutic uses thereof, particularly as vaccines.
In addition, the invention relates to the use of FHA for stimulating an immune response.
One of the objects of this invention is to expose the recombinant peptide at the surface of prokaryote cells, particularly for vaccination purposes. It should be noted that detoxification or attenuation of recombinant cells is only mentioned accessorily and that further neither an example nor a particular procedure illustrates this possibility. In particular, this invention does not mention accurately how the cells are detoxified or attenuated.
Thus, it results from the analysis of the prior art that vaccination means against pertussis have been known before, but that they induced side-effects.
The applicant had thus made efforts to find vaccination means against pertussis, but also against other pathogens, deprived of side-effects, easy to implement and able to be produced at low cost.
We have first found surprisingly that an intranasal administration of a strain deprived of B. pertussis toxin gene induced a high production of antibodies directed against the filamentous hemagglutinin (FHA) and that it was consequently possible to stimulate the immune response against a heterologous antigen fused with the FHA.
We have found on the other hand that the FHA integrated into liposomes containing other peptides having epitopes, improved the binding of these liposomes with the mucosas of the respiratory tract.
We have finally found that the FHA had generally immunostimulating properties.
The present invention relates thus first to a Bordetella strain deficient in the production of toxins and expressing a hybrid protein comprising at least one part of the filamentous hemagglutinin (FHA) and at least one part of a heterologous protein to FHA.
Advantageously the Bordetella strain has been made deficient in the production of toxins by elimination or deletion, at least partially, or by mutation, of the gene coding for the toxin so as to produce an inactive toxin. Such a gene may be particularly the gene coding for B. pertussis toxin or any protein having a structure or function similarity with such a toxin. It can be also the gene coding for hemolysin/adenylate cyclase toxin or the dermonecrotic toxin expressed by Bordetella strains or for proteins having structure or function similarities.
The Bordetella strain may be deficient in the production of one or various of these toxins.
Advantageously a heterologous protein consisting in a part of the hybrid protein comprises at least an epitope of a protein capable of being expressed by pathogens upon infections of the upper or lower respiratory tract.
Consequently said hybrid protein may comprise part of the protein FHA and part of the protein Sm28GST of Schistosoma masoni. This particular protein may be expressed into a strain of B. pertussis species and may be particularly the strain BPNX deposited under No. I-1770 on Oct. 8, 1996, in the national Collection of Microorganism Cultures of xe2x80x9cl""Institut Pasteurxe2x80x9d, 28, Rue du Docteur Roux, F-75724, Paris, Cedex 15, France.
A strain according to the present invention may be obtained by elimination of the gene of the toxin from the genome of a virulent strain expressing said hybrid protein, or by partial deletion or by mutation so as to produce an inactive toxin.
Elimination may be carried out by any method known to those skilled in the art and particularly by crossing the virulent strain with a mobilizing strain, then by selecting, through markers adapted according to the strains, cells having lost the toxin gene. Such a loss of capacity of the virulent strain to express the toxin results from a double event of a homologous recombination between the virulent strain and a plasmid of the mobilizing strain. A person skilled in the art may refer for the obtaining of attenuated strains to the method described by Antoine and Locht (1990, Infect, Immun., 58, 1518-1526).
The characteristics of the deficient strains in the production of toxins, so selected, may be checked with various techniques, in particular by Western-blot.
The virulent strains expressing the hybrid protein may be obtained with the techniques known to those skilled in the art and, in particular, may be obtained as described in the above-mentioned French patent application FR-94 04 661 the contents of which are included into the present invention by reference. The recombinant DNAs comprising on the one hand a sequence coding for a heterologous peptide and on the other hand a sequence coding for a part of FHA are obtained through the methods known to those skilled in the art, in particular as described in Example V of the French patent application FR-94 04 661. This example results in the fusion of the region 190-211 of glutathion-S-transferase of 28 kDa (Sm28GST) of Schistosoma mansoni, with the truncated FHA protein. The recombinant DNAs coding for the hybrid proteins are selected, the sequence thereof is checked according to methods known to those skilled in the art, then transferred into Bordetella cells.
The person skilled in the art may refer for the implementation of the present invention to general manuals relating to these techniques and in particular to the following manual: Maniatis et al., 1982, Molecular Cloning: Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y., USA, or one of its recent re-editions.
It will be understood that the present inventions is not limited to Bordetella pertussis stains, but is applied to any Bordetella species, in particular these infectious for humans, including B. parapertussis or B. bronchiseptica or also to Bordetella strains infectious for animals, particularly the Bordetella brotichiseptica infectious for dogs or pigs or the B. avium strains for birds.
The heterologous protein, the sequence of which is included into the hybrid protein, may be any antigenic protein sequence, especially Bordetella, Shigella, Neisseria, Borrellia antigens, diphtheric, tetanic or choleric toxins or toxoids, viral antigens including hepatitis B, hepatitis C, polvovirus or HIV, or parasitic antigens such as these of Plasmodium, Schistosomas or be toxoplamas. It may be also include an epitope of a protein capable of being expressed by pathogens upon mucosal infections or systemic infections.
Such strains may be used for vaccination of humans or animals against various pathogens. They may be especially administrated intranasally.
Besides, the present invention relates to drugs and vaccines containing such strains or to pharmaceutical compositions containing a pharmacologically efficient quantity of such strains, and optionally one or more pharmacologically compatible excipients.
Another object of the present invention comprises the use of such strains to produce a drug or a vaccine for treating affections of the upper or lower respiratory tract, mucosal infections or systemic infections.
It is to be noticed that the strains according to the present invention have surprising activities. In fact, as shown in the following examples, the strains expressing hybrid proteins the toxin gene of which has been eliminated, induce a well higher production of antibodies than that obtained with the parent virulent strain, when both strains are administrated intranasally. The intranasal administration, for example by nasal spraying, allows the elimination of the contamination dangers associated with an injection through the skin and also avoids the destruction of vaccines administrated orally in the acidic environment of the stomach.
The present invention further relates to liposomes comprising at least one part of the FHA protein as well as at least a heterologous protein to FHA protein.
Such a heterologous protein may be a protein comprising the above-mentioned hybrid protein. It may thus comprise at least one epitope of a protein liable to be expressed upon infections of the upper or lower respiratory tract. Such liposomes may be obtained in a way known to those skilled in the art, for example with a process comprising the steps of mixing the FHA, the heterologous protein and lipids, then rehydrating the lipid film. They may be used for therapeutic uses to produce drugs and vaccines for stimulating the immune response against a given epitope.
The present invention thus relates to drugs containing such liposomes as well as pharmaceutical compositions containing pharmacologically efficient quantities of such liposomes as well as, optionally, pharmacologically compatible excipients.
The binding of the FHA with the liposome surface has the advantage of targeting towards the mucosal tissue the administration of the heterologous protein. The FHA will have thus two actions: on the one hand a targeting action of the liposomes due to a particular adherence property with respect to mucosal tissues and on the other hand immunostimulating properties will allow to increase the immune response against antigens of the heterologous protein.
This second property of the FHA is another object of the present invention.
Consequently, the present invention relates to the use of FHA to produce a drug for stimulating immune responses. Preferably, the FHA is associated with a heterologous molecule having an antigenic determinant or epitope, the action of which it will potentialize.